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5.1.6.2.
3-Benzyloxy-20-hydroxy-30,4,40,50,60-penta-
tamethoxylchalcone (181 mg, 0.38 mmol) and phospho-
ric acid (50 mg), 61 mg of 40-benzyloxy-30,5,6,7,8-
pentamethoxyflavanone was obtained as a pale yellow
solid. ESI-MS m/z = 481 [MH+]. 40-Benzyloxy-
30,5,6,7,8-pentamethoxyflavanone (61 mg, 0.13 mmol)
was treated with DDQ (57 mg, 0.25 mmol) to obtain
40-benzyloxy-30,5,6,7,8-pentamethoxyflavone (38 mg).
ESI-MS m/z = 479 [MH+]. Upon hydrogenation of
40-benzyloxy-30,5,6,7,8-pentamethoxyflavone (38 mg),
40-hydroxy-30,5,6,7,8-pentamethoxyflavone (XV, 40-
demethylnobiletin, 12.5 mg) was obtained as a white so-
lid. ESI-MS m/z = 389 [MH+]; 1H NMR (300 MHz,
CDCl3): d 8.80 (br, 1H), 7.78 (s, 1H), 7.50 (d,
J = 8 Hz, 1H), 7.0 (d, J = 8 Hz, 1H), 6.70 (s, 1H),
4.12(s, 3H), 3.98 (s, 3H), 3.94 (s, 3H), 3.92 (s, 6H); UV
(acetonitrile and water): kmax = 250.5 and 336.5 nm.22
methoxylchalcone (XVII). A solution of 3-benzyloxy-
4-methoxy-benzaldehyde (218 mg, 0.90 mmol) and
2-hydroxy-3,4,5,6-tetramethoxyacetophenone (217 mg,
0.85 mmol) in 10 mL of 80% ethanol containing KOH
(2 g) was stirred at room temperature overnight. The
mixture was acidified with 20% HCl and then extracted
with ethyl acetate. The extract was washed with water
and brine, dried over MgSO4, and concentrated in
vacuo, and the residue was applied to silica gel flash
chromatography to give 192 mg of 3-benzyloxy-20-hy-
droxy-30,4,40,50,60-pentamethoxylchalcone as
a light
yellow solid; ESI-MS m/z = 481 [MH+] and H NMR
1
was compatible with literature value.21
5.1.6.3. 30-Benzyloxy-40,5,6,7,8-pentamethoxyflava-
none (XVIII). A solution of 3-benzyloxy-20-hydroxy-
30,4,40,50,60-pentamethoxylchalcone (192 mg) in ethanol
(10 mL) containing 50 mg H3PO4 was heated to reflux
for 16 h. Upon concentration, the resultant material
was mixed with water and extracted twice with ethyl ace-
tate (50· 2 mL). The combined organic layer was con-
centrated and purified by normal phase flash
chromatography and 81 mg of 30-benzyloxy-40,5,6,7,8-
pentamethoxyflavanone was obtained as a pale yellow
solid; ESI-MS m/z = 481 [MH+] and 1H NMR was com-
patible with literature value.21
5.1.8. 3040-Didemethylnobiletin (XIV). The same proce-
dure as the previous synthesis of 30-demethylnobiletin
(XV) was followed using 3,4-dibenzyloxy-benzaldehyde
(287 mg, 0.90 mmol) and 20-hydroxy-30,40,50,60-tetra-
methoxyacetophenone (XVIII, 217 mg, 0.85 mmol) as
starting materials. 3,4-Dibenzyloxy-20-hydroxy-30,40,
50,60-tetramethoxylchalcone (202 mg) was obtained as a
light yellow solid; ESI-MS m/z = 557 [MH+]. From 3,4-
dibenzyloxy-20-hydroxy-30,40,50,60-tetramethoxylchalcone
(202 mg, 0.36 mmol) and phosphoric acid (50 mg), 89 mg
(0.16 mmol) of 30,40-dibenzyloxy-5,6,7,8-tetramethoxyf-
lavanone was obtained as a yellow solid, which was
treated with DDQ (57 mg, 0.25 mmol) to obtain 30,40-dib-
enzyloxy-5,6,7,8-tetramethoxyflavone as an off-white so-
lid (53 mg) as an off-white solid. ESI-MS m/z = 555
[MH+]. Upon hydrogenation of 30,40-dibenzyloxy-
5,6,7,8-tetramethoxyflavone, 30,40-dihydroxy-5,6,7,8-tet-
ramethoxyflavone (30,40-didemethylnobiletin, XVI,
13 mg) was obtained as a white solid; ESI-MS m/z = 375
[MH+]; UV (max in acetonitrile/water) 368.5 and
5.1.6.4. 30-Benzyloxy-40,5,6,7,8-pentamethoxyflavone
(XIX). A solution of 30-benzyloxy-40,5,6,7,8-pentameth-
oxyflavanone (81 mg, 0.17 mmol) and 2,3-dichloro-5,6-
dicyanobenzoquinone (DDQ, 57 mg, 0.25 mmol) in
dry dioxane (5 mL) was refluxed for 8 h. The reaction
mixture was cooled, concentrated, and separated with
silica gel chromatography to obtain 48 mg of 30-benzyl-
oxy-40,5,6,7,8-pentamethoxyflavone as an off-white
solid; ESI-MS m/z = 479 [MH+].
1
252.5 nm; H NMR (CDCl3, 300 MHz) d 7.70 (s, 1H),
5.1.6.5. 30-Demethylnobiletin (XV). 30-Benzyloxy-
40,5,6,7,8-pentamethoxyflavanone (48 mg) was dissolved
in methanol (2 mL) and to this solution 10% of palladium
on carbon (5 mg) was added. Hydrogen gas was intro-
duced at 40 psi for 30 min while shaking to give 30-hydro-
xy-40,5,6,7,8-pentamethoxyflavone (30-demethylnobiletin,
7.50 (d, J = 8.5 Hz, 1H), 7.0 (d, J = 8.5 Hz, 1H), 6.74 (s,
1H), 4.12(s, 3H), 4.02 (s, 3H), 3.97 (s, 3H), 3.94 (s, 3H);
13C NMR (CDCl3, 75 MHz), d 178.4, 163.1, 152.4,
148.9, 148.4, 148.2, 145.0, 144.9, 139.0, 123.2, 119.6,
115.7, 114.8, 113.5, 106.3, 62.4, 62.3, 61.3, 61.8.
15 mg) as a white solid. ESI-MS m/z = 389 [MH+]; H
5.2. Biological activity
1
NMR (300 MHz, CDCl3): d 7.50 (d, J = 2 Hz, 1H), 7.48
(dd, J = 8.5 and 2 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H),
6.60 (s, 1H), 5.78 (s, 1H), 4.10(s, 3H), 4.02 (s, 3H), 3.98
(s, 3H), 3.94 (s, 6H); 13C NMR (75 MHz, CDCl3): d
177.52, 161.32, 159.80, 149.69, 148.24, 147.92, 146.35,
144.40, 138.80, 124.42, 118.98, 115.50, 112.49, 111.04,
107.26, 62.434, 62.22, 61.98, 61.81; UV (acetonitrile and
water): kmax = 250.5 and 374.5 nm.22
5.2.1. Cell culture. Human leukemia HL-60 cell lines were
obtained from American Type Culture Collection (Rock-
ville, MD) and maintained in Roswell Park Memorial
Institute 1640 medium (Life Technologies, Inc.) which
was supplemented with 10% fetal bovine serum (Biologi-
cal Industries, Israel), 2 mM glutamine (Gibco BRL) and
1% penicillin/streptomycin (10,000 units of penicillin/mL
and 10 mg/mL streptomycin). The HL-60 cell lines was
kept at 37 ꢁC in humidified 5% CO2 incubator.
5.1.7. 40-Demethylnobiletin (XIII).21,22 The same proce-
dure as the previous synthesis of 30-demethylnobiletin
(XV) was followed using 4-benzyloxy-3-methoxy-
benzaldehyde (218 mg, 0.90 mmol) and 20-hydroxy-
30,40,50,60-tetramethoxyacetophenone (217 mg, 0.85 mmol)
5.2.2. Cell proliferation assay. HL-60 cells were placed
into 12-well plates at a density of 2 · 105 per well. After
overnight growth, cells were treated with different con-
centrations of the selected compounds for 24 h. Control
cells are treated with DMSO to a final concentration of
0.05% (v/v). At the end of incubation, cells are harvested
and counted in a hemocytometer.
as
starting
materials.
4-Benzyloxy-20-hydroxy-
3,30,40,50,60-pentamethoxylchalcone (181 mg) was ob-
tained as a light yellow solid. ESI-MS m/z = 481
[MH+]. From 4-benzyloxy-20-hydroxy-3,30,40,50,60-pen-